EP3427036B2 - Method for a high-resolution local imaging of a structure in a sample in order to detect reactions of an object of interest to altered environmental conditions - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION

The invention relates to methods for high-resolution imaging of a structure marked with luminescence markers in a sample.

The invention falls in the field of high-resolution scanning luminescence light microscopy, in which measures are taken that allow luminescence light emitted from the respective sample with higher spatial resolution than the diffraction limit at the wavelength of the luminescence light and at the wavelength of any excitation light with which the luminescence markers spatially be limited to the emission of luminescent light to be assigned to a location in the sample. The luminescence markers are often fluorescence markers which, after being excited by excitation light, emit fluorescent light as luminescence light. Then one speaks of fluorescence microscopy.

STATE OF THE ART

In known methods and scanning luminescence light microscopes, to increase spatial resolution, light affecting the emission of luminescence light by the luminescence markers is directed onto the sample with an intensity distribution that has a zero point adjacent to intensity maxima. In many cases, this light is luminescence prevention light, with which the emission of luminescence light is prevented by those luminescence markers that are located outside the zero point. The luminescence light emitted from the sample can thus be assigned to the location of the zero point, because only luminescence markers arranged there are able to emit luminescence light.

In STED fluorescence microscopy, fluorescence markers previously excited with excitation light are de-excited again by means of stimulation light as fluorescence prevention light, except for those in the area of the zero point, by stimulated emission, so that only the fluorescence markers located in the area of the zero point can have emitted the subsequently measured fluorescence light. This fluorescent light can thus be assigned to the location of the zero point in the sample. The spatial distribution of the fluorescent markers in the sample is determined by scanning the sample with the zero. In this way, the structure and the spatial distribution of a structure marked with the fluorescent markers can be imaged in the sample.

In GSD fluorescence microscopy, those fluorescence markers outside the region of the zero point are converted to an electronic dark state with fluorescence prevention light and can thus not be excited by excitation light to emit fluorescence light.

RESOLFT fluorescence microscopy uses fluorescence-preventing light that converts photochromic fluorescent markers from a fluorescent state to a non-fluorescent state, except for those near the null. When the fluorescence markers are subsequently excited with excitation light, only the fluorescence markers in the region of the zero point of the intensity distribution of the fluorescence-preventing light are accordingly excited by the excitation light to emit fluorescent light. Here, too, the fluorescence light emitted by the fluorescence markers from the sample can be assigned to the location of the zero point of the intensity distribution of the fluorescence prevention light.

With all the methods of high-resolution scanning luminescence light microscopy described so far, there is a not inconsiderable risk of temporarily or even permanently bleaching the luminescence markers in the respective sample, i. H. to inactivate so that they no longer emit luminescent light. This danger is due to the fact that the intensity of the luminescence-inhibiting light in order to prevent all luminescent markers outside the area of the null from emitting luminescent light and at the same time to limit the spatial dimensions of the area of the null from which the luminescent markers can still emit luminescent light is strong narrow, must be set very high. The luminescence-inhibiting light acts on the luminescence markers in the sample with this high intensity already when the region of the zero point of the luminescence-inhibiting light approaches them, i. That is, even before they reach the area of the zero point for the first time and thus the luminescent light emitted by them is registered for the first time. The consequence of this can be that luminescence markers that tend to bleach cannot be used in the methods described, or at least not with very high intensities of the luminescence-preventing light, as would be desirable for maximizing the spatial resolution.

Various approaches have been pursued to counteract the problem of temporary and, in particular, permanent bleaching in high-resolution scanning luminescence light microscopy. the DE 10 2005 027 896 A1 teaches to apply the fluorescence prevention light in STED fluorescence microscopy in pulses with comparatively large time intervals or with rapid scanning of the respective sample with the zero point on the sample, so that the same areas of the sample are only exposed to the high intensity of the fluorescence prevention light in an optimized time repetition interval . In this way, the intensity of the fluorescent light available from the sample is increased because of the rate at which the fluorescent markers change from an excited intermediate state to a permanent or only slowly decaying dark state upon further excitation reach, is significantly reduced. In other words, with a relatively large repetition interval of applying the intensity distribution of the fluorescence preventing light to each area of the sample, the amount of fluorescence light obtainable from the entire sample in a certain period of time is maximized. This procedure also reduces the tendency of the fluorescence markers to bleach because a stronger population of excited states, from which photochemical destruction of the fluorescence markers occurs, is avoided.

To carry out high-resolution fluorescence microscopy with bleaching-prone fluorescent markers teaches DE 10 2011 051 086 A1 , Scanning conditions, which, in addition to a scanning speed at which the respective sample is scanned, and light intensities, an intensity distribution of fluorescence prevention light properties and a concentration of the fluorescence markers in the sample, to be coordinated in such a way that the fluorescence light in the form of individually detectable photons from the region of the zero point of the intensity distribution of the fluorescence prevention light is emitted. An image of a structure in the sample labeled with the fluorescent markers is then assembled from the locations assigned to the detected photons during several iterations of scanning the sample with the null. In this way, the probability of bleaching of the fluorescent markers is reduced before they are first reached by the zero point and thus detected. This is because the probability of bleaching is correlated with the intensity of the fluorescent light obtained from each fluorescent marker. Because the fluorescent light is minimized to single photons, the risk of bleaching is also minimized. Basically, the from the DE 10 2011 051 086 A1 However, according to the known methods, the individual fluorescence markers are only reached from the zero point of the fluorescence prevention light after they have previously been exposed to the high intensities in the region of the adjacent intensity maxima.

In order to be able to use substances that are sensitive to bleaching in high-resolution scanning luminescence light microscopy, it is WO 2011/131591 It is known to run a measurement front over a sample containing a structure of interest marked with luminescence markers. The intensities of optical signals over a depth of the measurement front that is smaller than the diffraction limit at the wavelength of the optical signals to such that a proportion of the luminescent markers that emit luminescent light by converting the luminescent markers into a luminescent state of not present increases and falls back to absent by converting the luminescent markers to a non-luminescent state. The luminescence light from the area of the measurement front is recorded and assigned to the associated positions of the measurement front in the sample. The assignment of the luminescence light along the measurement front can also be done with a spatial resolution beyond the diffraction limit, for example by assigning the registered photons to a single luminescence marker, as is done for example in a light microscopic method known as GSDIM.

One way to increase the speed of imaging a structure of interest of a sample in scanning luminescence light microscopy is to scan the sample with multiple nulls of the luminescence-inhibiting light in parallel. In this case, the luminescence light emitted from the sample is assigned separately to the individual zero points of the luminescence-preventing light. From the DE 10 2006 009 833 B4 it is known to form an intensity distribution of luminescence prevention light with a grid of zeros by superimposing two mutually orthogonal line patterns of luminescence prevention light in the sample. This prevents interference between the line patterns, so that their intensity distributions add up. The desired zeros of the intensity distribution of the luminescence-preventing light, which are adjacent to intensity maxima, remain at the crossing points of the line-shaped zeros of the two line gratings. In order to scan the sample completely in the area of the grid-like arrangement of the zeros, it is sufficient to shift each zero over the distances to the zeros next to it in the direction of both line patterns. Here, too, most of the luminescence markers in the sample are exposed to high light intensities of the luminescence prevention light before they are reached by the zero point and thus detected for the first time. The luminescent markers must therefore be chosen to withstand these high light intensities without fading.

Li D et al.: Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics, Science 2015 Aug 28; 349(6251 ) disclose a method for high-resolution imaging of a structure marked with activatable fluorescence markers in a sample, the sample being scanned successively in different directions with coincident linear or planar zero points of light intensity distributions of fluorescence activation light and fluorescence excitation light, and the fluorescence light emitted by the sample being scanned with a camera is detected. From the evaluation of the recorded light intensities, an image of a structure of interest in the sample can be reconstructed, the spatial resolution of which is increased by the narrowing of the matching zero points of the fluorescence activation light and the fluorescence excitation light, from which no fluorescence light originates from the sample. In this known method, too, the zero points of the fluorescence activation light and of the fluorescence excitation light acting at the same time as fluorescence deactivation light are of intensity maxima of the fluorescence activation light and the fluorescence excitation light. All luminescence markers in the sample are exposed to the high intensities of the fluorescence activation light and the fluorescence excitation light in the area of these intensity maxima before they reach the area of the coincident zero points of the fluorescence activation light and the fluorescence excitation light. With this known method, the risk of bleaching of the fluorescence markers before they contribute to the relevant measurement signal is therefore very high.

From the WO 2014/108455 A1 a method for spatially high-resolution imaging of a structure of a sample marked with luminescence markers is known, in which the sample is exposed to excitation light and stimulation light as luminescence prevention light, as in STED fluorescence microscopy, in order to surround the area of the sample, the fluorescent light emitted and detected from the sample can be assigned to the region of a null point of the stimulation light. In order to protect the luminescence markers from the high intensities of the stimulation light in the area of its intensity maxima adjacent to the zero point, the sample is additionally exposed to excitation-preventing light whose intensity distribution has a local minimum that coincides with the zero point of the stimulation light. This excitation prevention light can in particular be switch-off light, which switches switchable luminescence markers outside the minimum of the excitation prevention light into an inactive state in which they cannot be excited to emit luminescence light with the excitation light. In concrete terms, the luminescence markers can be switchable fluorescent dyes, such as are used in high-resolution RESOLFT fluorescence microscopy. At the one from the WO 2014/108455 known methods, the switchability of the luminescence markers is not primarily used to increase the spatial resolution, but to protect them from bleaching due to the high intensities of the stimulation light.

Out of RA Hoebe et al.: Controlled light-exposure microscopy reduces photobleaching and phototoxicity in fluorescence live-cell imaging, Nature Biotechnology, Volume 25, No. 2, February 2007, pages 249 to 253 a method of confocal fluorescence microscopy is known in which a sample is scanned with focused excitation light in order to image a structure in the sample marked with luminescence markers. The excitation light is switched off in each position of the focused excitation light in the sample as soon as a number of photons emitted by the excited luminescence markers in the sample and registered with a detector reaches an upper limit that corresponds to a desired signal-to-noise ratio is equivalent to. The excitation light is also switched off if the number of emitted and registered photons does not reach a lower limit within a specified partial period of the maximum pixel dwell time, because this indicates that there is no relevant concentration of luminescence markers at the respective position of the focused excitation light . In this way, the exposure of the sample to excitation light is significantly reduced compared to exposure of the sample to the same amount of light in each position of the focused excitation light.

Out of T. Staudt et al.: Far-field optical nanoscopy with reduced number of state transition cycles, Optics Express Vol. 19, No. 6, 2011 March 14, pages 5644 to 5657 a method called RESCue-STED is known, which is that of RA Hoebe et al. transfers the methods described for confocal fluorescence microscopy to STED fluorescence microscopy. As a result, the sample is only exposed to the high intensities of the stimulation light for as long as is necessary or expedient.

From the DE 10 2013 100 147 A1 discloses a method for spatially high-resolution imaging of a structure of a sample that has a luminophore, in which the sample is exposed in a measurement area to an intensity distribution of luminescence-inhibiting light that has a local minimum. Thereafter, the sample in the measurement area is exposed to luminescence excitation light, which excites the luminophore from an electronic ground state into a luminescent state; and luminescent light emitted from the measurement area is registered. The registered luminescence light is assigned to the position of the local minimum in the sample. The electronic ground state of the luminophore is disturbed with the luminescence-preventing light in such a way that the luminophore has a reduced absorption cross section for the luminescence excitation light in the disturbed electronic ground state. In addition, before registering the luminescence light in the measurement area, the sample can be exposed to STED light, which also has a local minimum in the center of the measurement area. If this local minimum is even smaller than the local minimum of the intensity distribution of the luminescence-preventing light, the spatial allocation of the luminescence light can thereby be even more limited, as a result of which the spatial resolution when imaging the structure is increased even further. The local minima of the STED light and the fluorescence prevention light are arranged concentrically with each other in all positions of the local minimum of the intensity distribution of the fluorescence prevention light in the sample.

From the DE 10 2013 114 860 B3 a method for determining the locations of individual molecules of a substance in a sample is known, in which the individual molecules of the substance are in a fluorescent state in which they can be excited with excitation light to emit fluorescent light, and the distances of the individual molecules of the substance a comply with the minimum value. The individual molecules of the substance are excited with excitation light to emit fluorescent light, with an intensity distribution of the approaching light having at least one zero point. The fluorescent light from the excited individual molecules of the substance is registered for different positions of the at least one zero of the intensity distribution of the excitation light. In this case, the distances between the most closely adjacent positions of the at least one zero point, in which the fluorescent light is registered by the excited individual molecules of the substance, are not greater than half the minimum value. The locations of the individual molecules of the substance are then derived from the progression of the intensity of the fluorescent light from the respective molecule over the positions of the at least one zero point of the intensity distribution of the excitation light in the region of interest of the sample.

From the WO 2010/069987 A1 discloses a method for the dynamic displacement of a light beam relative to an optical system that focuses the light beam. This method can be used in STED microscopy. When scanning the sample, fast adaptive raster patterns can be implemented that omit dark background areas or object areas that are uninteresting for other reasons, in order to achieve a higher image repetition rate through a reduced number of pixels.

the US 2012/0104279 A1 describes a fluorescence light scanning microscope with a birefringent chromatic beam shaping device. The scanning microscope is in particular a STED microscope. Both excitation light and STED light pass through the birefringent chromatic beam-shaping device, with the beam-shaping device not impairing the formation of an intensity maximum of the excitation light in the focus of a downstream objective, but having the result that the STED light has an intensity distribution with an intensity minimum locally of the maximum intensity of the excitation light.

From the EP 2 317 362 A1 a microscopic method with increased resolution is known in which a sample is scanned twice with an intensity maximum of excitation light in order to generate two images of the sample. The scanning points on which the two images are based are offset from one another by an increment below an optical resolution limit of the method, and the resulting differences in the images are evaluated to achieve increased spatial resolution. A corresponding method is also from the DE 10 2013 017 468 A1 known.

From the U.S. 2013/0201558 A1 a beam shaping device for shaping a beam with a central null point is known. This beam shaping device can be used in STED microscopy. In the beam shaper, a scan angle set when the beam is scanned across a sample is limited because it perturbs the relative delays of the beam set by various retardation plates to form the center null.

From the U.S. 7,679,741 B2 a method for the spatially high-resolution examination of samples is known, which belongs to RESOLFT microscopy with switchable substances. In one embodiment, the known method has the parallel formation of several zero points of fluorescence prevention light in order to examine the sample in parallel, ie simultaneously in several partial areas.

OBJECT OF THE INVENTION

It is the object of the invention to show methods for high-resolution imaging of a structure marked with luminescence markers in a sample and a scanning luminescence light microscope for carrying out such methods with which the exposure of the luminescent markers in the sample to high light intensities is fundamentally reduced, so that even compared to high light intensities sensitive luminescence markers can be used and a structure in the respective sample can be repeatedly imaged in order to record the reactions of an object of interest to changing environmental conditions.

SOLUTION

The object of the invention is achieved by methods according to the independent patent claims. The dependent claims relate to preferred embodiments of the first method according to the invention.

DESCRIPTION OF THE INVENTION

In a method according to the invention for high-resolution imaging of a structure marked with luminescence markers in a sample, light that affects the emission of luminescence light by the luminescence markers is directed onto the sample with an intensity distribution that has a zero point adjacent to intensity maxima. Sections of the sample to be scanned are scanned with the zero, and luminescent light emitted from the region of the zero is registered and assigned to the location of the zero in the sample. In this case, a plurality of specimens of an object of interest are each arranged so that they overlap with one of the partial regions of the sample to be scanned, and the multiple specimens of the object of interest are subjected to changed environmental conditions in order to record the reactions of the object of interest thereto. The individual sections of the sample are affected during and/or before and after the changed environmental conditions sampled at the respective zero point. Dimensions of the portions of the sample to be scanned are limited to no more than 75% of a spacing of the intensity maxima in at least one direction and preferably in any direction in which the null is bounded by the intensity maxima in that direction.

Insofar as luminescence markers are mentioned here, they can in particular be fluorescent markers. However, other luminescence markers can also be used whose luminescence properties are based, for example, on chemiluminescence or electroluminescence. This includes the fact that the excitation of the luminescence markers to emit luminescence light in the method according to the invention is not restricted to specific mechanisms. In many cases, however, the luminescence markers will be excited to emit luminescence light by excitation light.

The light that affects the emission of luminescence light by the luminescence markers can be luminescence prevention light, which prevents the emission of luminescence light by the luminescence markers, for example by converting the luminescence markers to a dark state or by de-exciting excited luminescence markers by stimulated emission and thus prevents the emission of luminescent light. However, the light that affects the emission of luminescent light by the luminescent markers can also be light that converts the luminescent markers from a non-luminescent state into another non-luminescent state in which they are particularly well protected from bleaching . Furthermore, the light that affects the emission of luminescence light by the luminescence markers can also be light that affects the emission of luminescence light by the luminescence markers not alone but only in conjunction with other light in such a way that these only still occurs from the area of the zero point of the intensity distribution of the light.

In another embodiment, the light that affects the emission of luminescence light by the luminescence markers is luminescence-enabling light that enables the emission of luminescence light by the luminescence markers in the first place, for example by stimulating the luminescence markers to luminescence, i.e. luminescence-excitation light , or converting the luminescent markers from a dark state to an excitable state.

The zero point of the intensity distribution of the light that affects the emission of luminescence light by the luminescence marker is at least a local intensity minimum of the light. There will often be an intensity minimum in which the intensity of the light essentially goes back to zero. Ideally, the intensity of the light actually goes to zero at the center of the null. However, this is not a mandatory requirement. If the light is, for example, luminescence prevention light, it is sufficient if the intensity of the luminescence prevention light in the region of the zero remains so low that it does not lead to any, or at least no significant, i. H. at least not leading to a predominant prevention of the luminescence of the luminescence markers. In this case, the zero point is delimited by the regions in which the luminescence-preventing light at least essentially prevents the emission of luminescence light by the luminescence markers. Everything between these areas, in which the luminescence-preventing light at least essentially prevents the emission of luminescent light by the luminescent markers, is referred to here as "null point" or "area of the null point".

If the intensity maxima, which are adjacent to the zero point of the intensity distribution of the light affecting the emission of luminescence light by the luminescence markers, are mentioned in the plural, this should not rule out the case that the zero point is, for example, ring-shaped the zero extending intensity maximum is enclosed. In each section through the intensity distribution of the light affecting the emission of luminescence light, such a ring-shaped intensity maximum is shown in the form of two intensity maxima, which are adjacent to the zero point in the section on both sides.

The null can be adjacent in one, two or three directions from the intensity maxima. It can therefore be a level, line or point zero point. The zero point can intersect a two-dimensional or one-dimensional sample in such a way that a line or point zero point in the dimensions of the sample is also adjacent to the intensity maxima on all sides. In addition, the partial areas of the sample to be scanned can be scanned in different directions with a zero point that is not adjacent to the intensity maxima in all dimensions of the sample, in order to maximize the spatial resolution when imaging in all directions. In the method according to the invention, the dimensions of the partial regions of the sample to be scanned are limited in at least one direction and preferably in all directions in which the zero point in the sample is adjacent to the intensity maxima.

The intensity maxima adjacent to the zero point in the sample are often of much higher light intensity than the areas of the intensity distribution of the light affecting the emission of luminescent light by the luminescent markers, which are directly adjacent to the zero point and in which the light is already reflected in the desired manner affects the emission of luminescent light by the luminescent markers, for example by preventing this emission. The very high intensities in the area of the maximum intensity are the result of the overall high intensity of the light, which in turn is a prerequisite for the region of the zero point in which the light has at least essentially no effect on the emission of luminescence light by the luminescence markers to be strongly spatially delimited. As a result, intermediate areas remain between the areas directly delimiting the zero point and the intensity maxima of the intensity distribution, in which the light intensity is significantly below the light intensity in the intensity maxima. These intermediate areas are used in a targeted manner in the method according to the invention, in that the dimensions of the scanned partial areas of the sample do not exceed 75% of the distance between the intensity maxima in this direction. If the dimensions of the scanned partial areas in the respective direction remain smaller than 50% of the spacing of the intensity maxima in the respective direction, no point of the scanned partial areas will be exposed to the full intensity of the light in the intensity maxima. However, even if the distance is limited to 75%, there is a significant limitation of the average intensity of the light that affects the emission of luminescent light and that is applied to the partial regions of the sample to be scanned. It goes without saying that the mean intensity of the light that is applied to the partial areas of the sample to be scanned decreases the more its dimensions remain below the distance between the intensity maxima in the respective direction. In absolute terms, the dimensions of the partial areas to be scanned in the respective direction between the intensity maxima can be a maximum of 300 nm, preferably a maximum of 200 nm and even more preferably about 100 nm. These absolute details relate to wavelengths of the luminescence light, the light affecting the emission of luminescence light by the luminescence markers and/or any excitation light in the visible range.

The sub-areas of the sample to be scanned can be sub-areas of the sample which, when the method according to the invention is carried out, alone, i. H. exclusively, and which are respectively aligned to regions of interest of the sample that overlap with the multiple instances of the object of interest.

With the method according to the invention, all specimens of the object of interest can in principle be subjected to the same changed environmental conditions. However, it is preferred to subject different subsets of the multiple specimens of the object of interest in the sample to different changed environmental conditions. In particular, this makes it possible to detect and compare different reactions of the object of interest to the different changed environmental conditions. The reactions can differ both in terms of the reaction result and the reaction speed. In this context, it has proven to be advantageous that the method according to the invention also enables faster repetitions of the scanning of the partial areas to be scanned with the zero point.

The environmental conditions to which the specimens of the object of interest are exposed in the method according to the invention can be different physical environmental conditions, for example different temperatures, different electric, magnetic and electromagnetic fields and the like. However, the environmental conditions can also be changed by adding a chemical substance, ie they can include different chemical environmental conditions. In a specific embodiment, the method according to the invention can be used to carry out drug screening, in which substances are searched for that cause the desired reactions in the object of interest. To this end, many specimens of the object of interest can be exposed individually or in larger subsets to different substances, which are examined for their suitability for eliciting the desired reaction. The high spatial resolution of the method according to the invention makes it possible, for example, to detect spatial changes in the object of interest as a result of the effect of the respective substance, which cannot be detected with many other methods or can only be detected indirectly and then with correspondingly greater effort.

For example, in order to carry out the drug screening referred to particularly efficiently, the plurality of specimens of the object of interest are arranged in a pattern defined in relation to fixed points of the sample in the second method according to the invention. It is then possible very efficiently to approach the sub-areas of the sample to be scanned with the zero point relative to the fixed points of the sample. This can also be done fully automatically. Arranging the objects of interest in the pattern can be done, for example, with the help of an immune reaction or the like.

In the method according to the invention, the partial areas of the sample to be scanned are usually scanned repeatedly with the zero point in order to record the reactions of the object of interest to the changing environmental conditions. It is preferred, at least when the scanning of the partial areas of the sample to be scanned is repeated, to arrange the zero point at no more than 3n or even no more than 2n locations per partial area to be scanned, where n is the number of spatial directions in which the partial areas of the sample to be scanned Sample to be scanned to image the object of interest with high resolution. Such a few null locations are sufficient to detect the location of individual luminescent markers in each sub-area to be scanned, if that is for each sub-area to be scanned registered luminescence light can each be assigned to a specific one of these luminescence markers. Such an assignment is possible if at any time only one of the luminescence markers emits the luminescence light, ie a temporal distinction is possible, or if the luminescence light can be assigned to individual luminescence markers based on its wavelength, for example. This embodiment of the method according to the invention thus makes use of the method known as MINFLUX. In MINFLUX, to determine the location of an individual luminescence marker in a plane, a zero of a light intensity distribution of excitation light is positioned at four different locations in the sample and the intensity of the luminescence light from the luminescence marker is recorded for this purpose.

The object of interest, which is arranged in multiple copies in the sample, can be a molecule, for example a protein molecule, a complex, but also a more complex biological entity such as a synapse, a membrane, another cell component or a whole virus act.

The sequence of the successive scans of the partial areas with the respective zero point can be very fast, so that rapid changes in the object of interest can also be detected.

The invention consciously accepts that the sub-areas of the sample that are scanned and thus imaged remain very small. Often they extend a distance of the order of the diffraction limit at the wavelength of light affecting the emission of luminescent light by the luminescent markers. Due to the significant reduction in the average light intensities affecting the scanned sub-areas, however, it is possible to successfully use luminescence markers that have a relatively strong tendency to bleach or to repeatedly scan the scanned sub-areas of the sample with the zero point of the intensity distribution.

The possibility of quickly and repeatedly scanning the sample in the partial areas with the zero point of the intensity distribution also makes it possible to resolve dynamic processes in the structure of interest in the sample over time. Since the luminescence markers in the sample have a particularly low tendency to bleach as a result of the method according to the invention, i.e. a particularly large number of photons are obtained from each individual fluorescence marker in each partial area of the sample to be scanned, a particularly large number of images of each partial area of the sample to be scanned can be recorded and thus longer-term changes in the structure of interest can be observed in the sample. As a rule, a partial area of the sample is scanned in no more than 100×100=10,000 pixels in the method according to the invention. This is possible in a few milliseconds. This allows image frequencies of 100 Hz and more to be realized.

In the method according to the invention, a partial area of the sample to be scanned can also be scanned with only very few positions of the zero point in the sample, for example to detect the displacement of an individual fluorescent marker in the sample due to changed environmental conditions.

Advantageously, the dimensions of the partial areas of the sample to be scanned are not greater than 45%, 25% or even 10% of the distance between the intensity maxima in each direction in which the intensity maxima are adjacent to the zero point in the sample. In relation to the intensity of the light affecting the emission of luminescence light by the luminescence marker, it is advantageous if the dimensions of the partial areas of the sample to be scanned are not greater than the distance in each direction in which the intensity maxima are adjacent to the zero point in the sample , over which the intensity of the light increases from the zero point in the respective direction to 50%, 25%, 10% or 5% of the intensity of the light in the adjacent intensity maxima. Correspondingly, the maximum exposure of the luminescence markers in the area to be scanned is then also limited to 50%, 25%, 10% or 5% of the intensity of the light in the adjacent intensity maxima.

In the method according to the invention, it often makes sense to image the specimens of the object of interest in the sample marked with the luminescence markers in a different way before scanning the partial areas of the sample to be scanned with the zero point, in order to determine the position of the partial areas of the sample to be scanned. The sub-areas of the sample to be scanned are those sub-areas of the sample that overlap with the specimens of the object of interest in such a way that particular details of the object or also developments of the object due to the changed environmental conditions appear in them. This imaging can take place with local or large-area excitation of the luminescence markers to emit luminescence light.

Before scanning the partial areas of the sample to be scanned, a larger area of the sample with the zero point can also be scanned with an intensity of the light affecting the emission of luminescent light that is at least 50% lower and/or with a scanning speed that is at least 50% higher in order to determine the position of the sub-areas of the sample to be scanned. In this preceding scanning, all points of the larger area of the sample are admittedly exposed to the high intensity of the light in the area of the intensity maxima. However, this intensity is deliberately reduced and/or it only acts on the luminescence markers over a shorter period of time.

In one embodiment of the method according to the invention, different scanning means are used for scanning a larger area of the sample than for scanning the individual partial areas of the sample to be scanned Sample.

If different scanning means are used to scan the larger area of the sample in order to define the sub-areas of the sample to be scanned, and for the subsequent scanning of the sub-areas of the sample to be scanned, scanning means that are specially tailored to the scanning of the individual, narrowly defined sub-areas of the sample to be scanned can be used. Due to the small dimensions of the sub-areas in the sample to be scanned, these can be scanning means that do not allow large shifts in the light intensity distribution with the zero point, but that convert the shifts that they can cover very quickly and/or precisely. In this way, the partial areas to be scanned can be scanned very quickly, for example in order to track rapid changes in the objects of interest in the partial areas.

Specifically, to scan the larger area of the sample in at least one direction, a sample holder for the sample can be moved relative to a lens with which the light is directed onto the sample, while an electro -optical scanner, an acousto-optical deflector or a galvo mirror, i.e. a deflecting mirror with galvanometer drive is used. The device for scanning the individual partial areas of the sample to be scanned can be combined with an additional electro-optical or acousto-optical modulator as a phase shifter for shifting the zero point of the light intensity distribution.

As already mentioned, the light affecting the emission of luminescence light by the luminescence markers can be, in particular, luminescence prevention light, which prevents the emission of luminescence light by the luminescence markers outside the zero point. For example, the luminescence-preventing light converts or switches luminescent markers in the form of switchable proteins into a dark state in which they cannot be excited to emit luminescent light. The luminescence prevention light can be directed onto the sample, in particular together with excitation light, which excites the luminescence markers to emit luminescence light and which has an intensity distribution with an intensity maximum in the region of the zero point of the luminescence prevention light. Except for the narrow limits for the sub-areas of the sample to be scanned, this corresponds to the usual procedure in STED, RESOLFT or GSD fluorescence light microscopy.

In the first method according to the invention is from the WO 2014/108455 A1 known concept of performing STED fluorescence microscopes with switchable luminescence markers in order to protect the luminescence markers from the high intensities in the region of the intensity maxima of the stimulation light by switching them to an inactive state, applied in a modified form. Specifically, before scanning the sub-areas of the sample to be scanned with the zero, additional switch-off light with such an intensity distribution is directed onto the sample that it switches the switchable luminescence markers in sub-areas of the sample adjoining the sub-areas to be scanned into an inactive state. These adjoining sub-areas adjoin the sub-areas to be scanned in the at least one direction in which the intensity maxima are adjacent to the zero point of the stimulation light in the sample. The luminescence markers are thus switched to the inactive state where the intensity maxima of the stimulation light occur and where without this protective measure the luminescence markers would be lost due to the high intensities of the stimulation light when scanning the partial areas of the sample to be scanned. Since the bleaching of the luminescence markers is omitted in this embodiment of the method according to the invention, it can also be carried out successively for partial regions to be scanned that are directly adjacent to one another or even overlap one another. In other words, the sample can be scanned with the partial areas to be scanned, the partial areas to be scanned being in turn scanned with the zero point at all or at least at selected positions in the sample.

The intensity distribution of the switch-off light can have a local intensity minimum formed by destructive interference in the sub-area to be scanned subsequently, in which it at least essentially does not switch off the luminescence markers, i. i.e. by leaving the luminescence markers at least essentially in their active state, in which they can be excited by the excitation light. Depending on the selection of the switchable luminescence markers, this active state may require or at least make it useful that before and/or with a time overlap with the direction of the turn-off light onto the sample, turn-on light is directed onto the respective sub-area of the sample to be scanned, which switches the switchable luminescence markers into their active state turns on.

When switched on and/or off, switchable luminescence markers often also emit luminescence light. This luminescent light can be registered and evaluated in the method according to the invention. The aim of this evaluation can be, for example, a decision as to whether the sub-area to be scanned, which is delimited by the respective adjacent sub-area, is even scanned with the zero point or whether it is only exposed to excitation light in its entirety, with the luminescence light then emitted being registered confocally, or whether it is not at all is applied with excitation light because the low intensity of the luminescence light registered when switching on and / or off indicates that no relevant concentration of luminescence markers in the respective part to be scanned is present. The evaluation can also have the aim of determining under which conditions the directing of the stimulation light onto the sample in each position of the zero point in the sub-areas to be scanned and the registration of the luminescence light emitted from the area of the zero point is meaningfully broken off. This means that, for example, an upper and/or lower limit value for carrying out a RESCue method in the respective sub-area to be scanned can be defined as a function of the result of the evaluation.

In a special embodiment of the method according to the invention, luminescence light emitted from the area of the zero point is registered with a point sensor whose position relative to the sample is not changed when scanning the respective partial area of the sample to be scanned. That is, when registering the luminescent light with the point sensor, the change in position of the zero point of the light intensity distribution of the light affecting the emission of luminescent light by the luminescent markers is not taken into account. This is possible because the dimensions of the partial areas to be scanned are regularly smaller than the detection area of a point sensor in relation to the sample. This also applies to a point sensor arranged confocally to the center point of the respective partial area to be scanned. The luminescent light from the entire partial area of the sample to be scanned regularly even reaches this, because its dimensions are regularly below the diffraction limit at the wavelength of the luminescent light. The positionally fixed point sensor means that the zero point for scanning the respective partial area of the sample to be scanned is shifted by acting solely on the light affecting the emission of luminescence light by the luminescence markers. Any excitation light does not have to be shifted together with the light affecting the emission of luminescence light by the luminescence marker, since its intensity maximum also typically extends over the entire partial area of the sample to be scanned.

As has also already been mentioned, the light affecting the emission of luminescence light by the luminescence markers can alternatively be luminescence-enabling light, which enables the emission of luminescence light by the luminescence markers outside the zero point in the first place. This implies that the light is luminescence excitation light as the only light that is directed onto the sample. It also includes that the light is luminescent activation light, which converts the luminescent markers from a dark state to a luminescent excitable state, ie. H. activated. The light affecting the emission of luminescent light by the luminescent markers can also have both functions, i. H. of activation and excitation, and also have two components of different wavelengths. If a sub-area of the sample with the zero point of the intensity distribution of the light that affects the emission of luminescence light by the luminescence markers is scanned, this sub-area is kept small according to the invention so that the luminescence markers located in the sub-area are not involved, or at least as little as possible to apply the high light intensities in the area of the intensity maxima adjacent to the zero point. In this embodiment of the method according to the invention, the luminescence light emitted by the luminescence markers in the sample is typically detected with a camera, and the evaluation is carried out by unfolding the registered intensity distributions with regard to the current position of the zero point in the sample and the associated change in the intensity distribution of the luminescent light emitted from the sample and captured by the camera.

If only one luminescence marker emits the detected luminescence light from the sample in the partial areas to be scanned, its position in the sample can also be determined very easily from the luminescence light registered for the various locations of the zero point, for example by fitting a function having a local extremum, where the location of the extremum of the fitted function is equated to the position of the luminescence marker sought. This procedure is possible both when the light that affects the emission of luminescence light by the luminescence markers and whose intensity distribution has the zero point is luminescence-preventing light and when it is luminescence-enabling light or excitation light. In the first case, the intensity maxima of the luminescence prevention light adjacent to the zero point advantageously prevent luminescence light emitted by other luminescence markers in the vicinity from interfering with the position determination. In the second case, the respective luminescence marker in the partial area to be scanned is only exposed to a minimal amount of light and is therefore only minimally photochemically stressed.

The disadvantage of the method according to the invention, that the sampled and thus imaged partial area remains very small, can be at least partially compensated for by the sample being scanned in several partial areas at the same time. In particular, a grid of zero points of the light affecting the emission of luminescence light by the luminescence marker can be used, as is fundamentally known. According to the invention, however, the grid of the zero points is not shifted so far that the sample is imaged as a whole, ie over the full grid dimension. Rather, the individual partial areas in which the sample is scanned also remain in this embodiment of the invention procedures separately. This is the only way to reduce the risk of bleaching of the luminescence markers in the scanned partial areas of the sample according to the invention. It goes without saying that if this parallelized embodiment of the invention is carried out with switchable luminescence markers, which are only in their fluorescent state in the partial areas of the sample to be scanned, the sample can also be scanned with the partial areas to be scanned in order to image them completely .

Since, in the method according to the invention, a plurality of specimens of the object of interest are arranged in each case overlapping with one of the plurality of partial areas of the sample to be scanned, a partial image of this object is obtained when each partial area is scanned. If these partial images are statistically distributed over the object and their number is large enough, an image of the entire object of interest can be reconstructed from them. It goes without saying that this assumes that the different specimens of the object of interest are at least largely the same. In order to allocate the partial images to specific points of the object of interest, the specimens of the object of interest in the sample can also be imaged in a different way in order to determine their position and orientation relative to the scanned partial areas of the sample.

A scanning luminescence light microscope for carrying out the method according to the invention has a light source for the light which affects the emission of luminescence light by the luminescence markers, light shaping means which direct the light with the intensity distribution onto the sample which has the zero point adjacent to the intensity maxima, scanning means, in order to scan a partial area of the sample to be scanned with the zero point, a detector which registers luminescence light emitted from the area of the zero point, and a controller for carrying out the method according to the invention.

The detector may be a point detector, where the location of the point detector relative to the sample when scanning the sample portion to be scanned may be fixed, i. H. can detect the luminescence light emitted from the sample without it being descanned, because the partial area to be scanned usually has dimensions below the diffraction limit. Further scanning means can then be present, which differ from the scanning means for scanning the partial area of the sample to be scanned with the zero point and are provided for scanning a larger area of the sample.

Here, too, the scanning means for scanning the larger area of the sample in at least one direction can have a sample holder that can be moved relative to an objective of the light-shaping means, while the scanning means for scanning the partial area of the sample to be scanned in at least one direction have an electro-optical scanner, an acoustic optical deflector or a galvo mirror.

However, the detector can also be a point detector, for example, which captures the unscanned luminescence light from the sample, or a flat detector, such as a camera, which captures the unscanned luminescence light from a fixed position relative to the sample.

In a scanning luminescence light microscope for carrying out a STED method according to the invention, the light provided by the light source is stimulating light, and a further light source is provided for stimulating light, with the light-shaping means directing the stimulating light onto the sample with a light intensity distribution which has a maximum in the region of the zero point of the Having luminescence prevention light.

To carry out the method according to the invention, which makes use of switchable luminescence markers, a switch-off light source for switch-off light must also be provided in the scanning luminescence light microscope, with the light-shaping means directing the switch-off light onto the sample with such an intensity distribution that the switchable luminescence markers are in a sub-area adjacent to the area to be scanned portion of the sample turns off to an inactive state. The adjoining sub-area borders on the sub-area to be scanned in the at least one direction in which the intensity maxima are adjacent to the zero point of the stimulation light in the sample. In addition, a switch-on light source can be provided for switch-on light, which switches the switchable luminescence markers on to their active state, with the light-shaping means directing the switch-on light to a sub-area of the sample that encompasses the sub-area to be scanned before and/or with a temporal overlap with the direction of the switch-off light onto the sample.

In experimental tests of the method according to the invention, an increase in the yield of the photons from the luminescence markers in the sample by a factor of >100 was achieved. This z. B. means that 100 times more images of a changing structure of interest can be taken to document the changes. In addition, less time is required for each individual image because more and more photons per unit of time are emitted by the luminescence markers in the sample.

Advantageous developments of the invention result from the patent claims, the description and the drawings. The advantages of features and combinations of several features mentioned in the description are merely exemplary and can have an effect alternatively or cumulatively without the advantages necessarily having to be achieved by embodiments according to the invention.

The reference signs contained in the claims do not constitute a limitation of the scope of the subject matter protected by the claims They only serve the purpose of making the patent claims easier to understand.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1

zeigt schematisch Intensitätsverteilungen von Anregungslicht und von Fluoreszenzverhinderungslicht als Beispiel für Licht, das sich auf die Emission von Lumineszenzlicht durch in einer Probe angeordnete Lumineszenzmarker auswirkt, sowie die resultierende effektive Anregung von Fluoreszenzmarkern in einer Probe zur Emission von Fluoreszenzlicht.

Fig. 2

zeigt für die Intensitätsverteilung des Fluoreszenzverhinderungslichts gemäß Fig. 1 Abmessungen eines mit den Intensitätsverteilungen gemäß Fig. 1 bei einem erfindungsgemäßen Verfahren abzutastenden Teilbereichs einer Probe.

Fig. 3

ist eine schematische Darstellung eines abzutastenden Teilbereichs einer Probe in einer Draufsicht, wobei das Abtasten längs einer mäanderförmigen Bahn erfolgt.

Fig. 4

ist eine schematische Darstellung eines abzutastenden Teilbereichs einer Probe in einer Draufsicht, wobei das Abtasten längs einer Spiralbahn erfolgt.

Fig. 5

illustriert schematisch ein Fluoreszenzmikroskop als Beispiel für ein Rasterlumineszenzlichtmikroskop zur Durchführung des erfindungsgemäßen Verfahrens.

Fig. 6

zeigt (a) ein vorab aufgenommenes Konfokalbild einer Probe und (b) ein Teilbild der Probe, das erfindungsgemäß aufgenommen wurde, nachdem einzelne abzutastende Teilbereiche der Probe anhand des Konfokalbilds ausgewählt wurden.

Fig. 7

zeigt die Abhängigkeit zwischen der Anzahl der aufnehmbaren Bilder und den Abmessungen eines abgetasteten Teilbereichs der Probe.

Fig. 8

zeigt schematisch ein anderes Fluoreszenzmikroskop als in Fig. 5 als weiteres Beispiel für ein Rasterlumineszenzlichtmikroskop zur Durchführung des erfindungsgemäßen Verfahrens.

Fig. 9

ist eine schematische Darstellung eines abzutastenden Teilbereichs einer Probe bei gleichzeitiger Darstellung eines angrenzenden Teilbereichs, in dem mit dem Rasterfluoreszenzmikroskop Ausschaltlicht auf die Probe gerichtet wird.

Fig. 10

illustriert eine Anordnung mehrerer abzutastender Teilbereiche gemäß Fig. 9 in der Probe, um diese mit den abzutastenden Teilbereichen abzutasten.

Fig. 11

illustriert eine andere Anordnung abzutastender Teilbereiche in der Probe.

Fig. 12

ist ein Blockdiagramm zu einer Ausführungsform des erfindungsgemäßen Verfahrens.

Fig. 13

illustriert die Anordnung von abzutastenden Teilbereichen in einer Probe bei dem erfindungsgemäßen Verfahren gemäß Fig. 12.

Fig. 14

illustriert schematisch eine Reaktion eines interessierenden Objekts auf sich verändernde Umgebungsbedingungen; und

Fig. 15

erläutert die Anordnung einer Nullstelle an nur wenigen Orten der Probe beim Antasten eines abzutastenden Teilbereichs.

The invention is further explained and described below with reference to preferred exemplary embodiments illustrated in the figures.

1

FIG. 12 schematically shows intensity distributions of excitation light and fluorescence-preventing light as an example of light affecting emission of luminescence by luminescence markers arranged in a sample and the resulting effective excitation of fluorescent markers in a sample to emit fluorescent light.

2

shows for the intensity distribution of the fluorescence prevention light according to FIG 1 Dimensions of one with the intensity distributions according to 1 part of a sample to be scanned in a method according to the invention.

3

FIG. 12 is a schematic representation of a partial area of a sample to be scanned in a plan view, with the scanning taking place along a meandering path.

4

FIG. 12 is a schematic representation of a partial area of a sample to be scanned in a plan view, the scanning taking place along a spiral path.

figure 5

schematically illustrates a fluorescence microscope as an example of a scanning luminescence light microscope for carrying out the method according to the invention.

6

shows (a) a previously recorded confocal image of a sample and (b) a partial image of the sample that was recorded according to the invention after individual partial areas of the sample to be scanned were selected using the confocal image.

7

shows the relationship between the number of images that can be recorded and the dimensions of a scanned partial area of the sample.

8

shows a different fluorescence microscope than in FIG figure 5 as a further example of a scanning luminescence light microscope for carrying out the method according to the invention.

9

is a schematic representation of a partial area of a sample to be scanned with a simultaneous representation of an adjacent partial area in which switched-off light is directed onto the sample with the scanning fluorescence microscope.

10

illustrates an arrangement of several partial areas to be scanned according to FIG 9 in the sample in order to scan it with the partial areas to be scanned.

11

illustrates a different arrangement of sub-areas in the sample to be scanned.

12

is a block diagram of an embodiment of the method according to the invention.

13

illustrates the arrangement of partial areas to be scanned in a sample in accordance with the method according to the invention 12 .

14

schematically illustrates a response of an object of interest to changing environmental conditions; and

15

explains the arrangement of a zero at only a few locations on the sample when probing a sub-area to be scanned.

FIGURE DESCRIPTION

In 1 a section through an intensity distribution of excitation light 1 is shown above. The excitation light 1 has an intensity maximum 27 with maximum intensity I at a geometric focal point F. However, the intensity I is distributed over an area that extends far beyond the focal point F on all sides, the diameter of which corresponds to the diffraction limit at the wavelength lambda of the excitation light 1 and the numerical aperture NA of the lens used to focus the excitation light 1 into the focal point F according to lambda /NA corresponds. In order to limit the excitation of fluorescence markers in a sample achieved with the excitation light 1 to a smaller area than the extent of the intensity distribution of the excitation light 1, fluorescence prevention light 2 is additionally directed onto the sample, which has a zero point 4 adjacent to intensity maxima 3 . The fluorescence prevention light 2 prevents the emission of fluorescence light by fluorescence markers excited by the excitation light 1, for example in that the fluorescence markers are de-excited again by stimulated emission. Everywhere outside the region 5 of the null point 4 of the fluorescence prevention light 2, the intensity I of the fluorescence prevention light 2 is so great that this de-excitation occurs completely, that is, the ones arranged there Fluorescent markers do not emit fluorescent light. Conversely, "zero point 4" here means the entire area 5 within which the intensity I of the fluorescence prevention light 2 remains so small that it does not prevent the emission of fluorescence light by fluorescence markers arranged therein. 1 below shows the spatial distribution of the effective fluorescence excitation 6. This is limited to the area 5 of the zero point 4. If a sample is scanned with the zero point 4, fluorescent light emitted from the sample always comes from the area 5 and can be assigned to the sample according to this location.

When the zero point 4 approaches a structure of interest marked with a fluorescent dye when scanning a sample, the fluorescent markers first reach the area of the intensity maxima 3 and the intensities of the excitation light 1 superimposed thereon before they reach the area 5 of the zero point 4. In particular, when the sample is scanned line by line, the fluorescent markers are repeatedly exposed to high light intensities before fluorescent light is registered by them for the first time. This can lead to the fluorescent markers already being bleached before they are reached by the zero point 4 for the first time. Repeated scanning of the same sample, for example to observe changes over time in the structure of interest in the sample marked with fluorescent markers, is often impossible due to this effect.

However, if the area of the sample scanned with the zero point 4 is at most 3/4 or 75% of a distance D ₀ of the intensity maxima 3, as is shown in 2 is drawn in, is limited, the average load on the fluorescence markers in the scanned area, which is caused by the high intensities of the fluorescence prevention light 2 in the area of the intensity maxima 3, in particular in connection with the intensity of the excitation light 1, decreases 1 occurs. This burden decreases further if the dimensions of the area to be scanned are restricted to half the distance D ₀ of the intensity maxima 3 and less. If the dimensions are limited to less than D ₀ /2, no point of this sub-area comes into the immediate area of the intensity maxima 3 when scanning the partial area of the sample to be scanned. If the dimensions of the area to be scanned are restricted to Do/4, the maximum intensity is of the fluorescence prevention light 2 applied to the sample within the area to be scanned is limited to about I ₀ /2, where I ₀ is the maximum intensity of the fluorescence prevention light 2 in the intensity maxima 3.

3 illustrates the scanning of a partial area 7 to be scanned of a sample 8 shown as a detail with the area 5 of the zero point 4 along a meandering path 9 here corresponds to a center point 10 of the partial area 7 to be scanned. The intensity maxima run accordingly, or to put it better, the ring-shaped intensity maximum 3 here runs around the in 3 illustrated position of the zero point 4 still through the scanned portion 7, which is indicated by a dashed line 11. However, this overlapping can be prevented by further limiting the partial area 7 to be scanned to less than D ₀ /2. However, the limitation of the dimensions of the sub-area 7 to be scanned to approximately 2D ₀ /3 given here also achieves a considerable reduction in the mean exposure of the sample 8 in the sub-area 7 to the fluorescence prevention light 2 .

4 illustrates a partial area 7 of the sample 8 to be scanned that is limited to D ₀ /2. Here, the course of the ring-shaped maximum intensity 3 indicated by the dashed line 11 no longer reaches the partial area 7 at any position of the zero point 4 in the partial area 7 to be scanned 4 a spiral path 12 along which the partial area 7 to be scanned is scanned starting from the center point 10 . Irrespective of the shape of the path on which the zero point 4 is moved when scanning the partial area 7 to be scanned, the fluorescent light emitted from the sample 8 and registered is assigned to the respective location of the zero point 4 in the sample.

figure 5 1 illustrates a scanning fluorescence light microscope 13 which is particularly suitable for carrying out a method according to the invention. The scanning fluorescence light microscope 13 has a light source 14 for the fluorescence prevention light 2, the cross section of which is expanded with expansion optics 15 and the wave fronts of which are modulated over its cross section with the aid of a phase plate 16 in such a way that, when focusing with an objective 45, it is reflected in the sample 8 Zero point 4 with adjacent intensity maxima 3 according to the 1 and 2 forms around the respective focal point F. A further light source 17 with expansion optics 18 is provided for the excitation light 1 . The excitation light 1 and the fluorescence prevention light 2 are combined with a dichroic mirror 19, so that the excitation light 1 in the region 5 of the zero point 4 of the fluorescence prevention light 2 has its intensity maximum 27 according to 1 having. The fluorescent light 20 emitted from the sample, specifically the area of the zero point of the fluorescence prevention light 2, is coupled out with a dichroic mirror 26 and registered with a point detector 21 and assigned to the respective location of the zero point in the sample. For scanning the respective partial area of the sample with the zero point, scanning means 22 and 23 are provided for two mutually orthogonal scanning directions, which are controlled in a coordinated manner. The scanning means 22 and 23 influence only the direction of the excitation light 1 and the fluorescence prevention light 2, they could even exclusively in the optical path of the fluorescence prevention light 2 be arranged. Since the portion 7 of the sample 8 to be scanned has dimensions below the diffraction limit, even if the point detector 21 is stationary relative to the sample 8, the fluorescent light 20 emitted from the sample 8 from the area of the zero point of the fluorescence prevention light 2 always reaches the point detector 21, i.e. despite the displacement of the zero point using the scanning means 22 and 23. For a scanning of the sample 8 that goes beyond the partial area 7 of the sample 8 to be scanned, which is scanned according to the invention, in order, for example, to initially determine the position of a suitable partial area to be scanned, further scanning means are in the area a sample holder 24 is provided, which is indicated here only by corresponding displacement symbols 25. In the previous description, it was not explicitly stated that the zero point 4 of the intensity distribution of the fluorescence prevention light 2 can also be limited by intensity maxima 3 in a z-direction, in which the fluorescence prevention light 2 is directed onto the sample, in order to improve the spatial resolution during imaging of the structure of interest in the sample 8 also in this z-direction. Correspondingly, the partial area 7 to be scanned must then also be limited in this z-direction to a maximum of 75%, preferably less than 50% of the distance between the intensity maxima of the light in this direction, or also in the z-direction starting at its center point 10 and increasing the distance to scan to this center 10. An increased spatial resolution when imaging the structure in the z-direction can also be achieved in the method according to the invention by other measures, for example by a 4Pi arrangement or by 2-photon excitation of the fluorescent markers to emit the fluorescent light or by others in the field of Fluorescence microscopy known measures. It also applies in principle that the methods described here can be supplemented by other measures known in the field of fluorescence microscopy. This includes, for example, the application of the fluorescence prevention light 2 and/or the excitation light 1 in pulses, the simultaneous continuous application of the excitation light 1 or the fluorescence prevention light 2, the gated registration of the fluorescence light in a defined time window after the respective pulses, etc.

The confocal image according to Figure 6(a) was recorded from a sample in which a structure of interest was tagged with the luminescent marker nucleoporin gp210. The confocal image gives an overview of the structure of interest. Individual partial areas of the sample were selected from this overview, in which STED images were recorded using the method according to the invention. These partial areas are smaller than the focus of the excitation light. In the sub-areas, the structure of interest is imaged with high spatial resolution and at the same time high light yield. To record the in Figure 6(b) In the partial image of the sample shown, which reproduces the scanned partial areas, excitation light with a wavelength of 635 nm and a power of 5 μW was directed onto the sample in pulses with a repetition rate of 20 MHz. STED light with a wavelength of 775 nm was directed onto the sample in synchronized pulses with a pulse length of 1.2 ns and a power of 150 mW. The excitation light and the STED light were focused into the sample with a 1.4 NA oil inversion objective. The fluorescence light was focused onto a point detector using the oil inversion objective and another lens.

7 shows the bleaching behavior depending on the dimensions of the scanned part of a sample using the example of stained nuclear pore proteins in STED scanning fluorescence light microscopy. τ1/2 denotes the number of images that can be recorded until the fluorescence signal has fallen to half of the original value due to bleaching. τ1/2 is plotted against the dimensions of the scanned portion of the sample in nanometers. The STED power was 160 mW, the excitation power 2 µW. Otherwise the STED conditions corresponded to those according to 6 . With dimensions of the scanned partial area of 100×100 nm ² the bleaching is reduced by a factor of 100 compared to dimensions of 800×800 nm ² . Accordingly, 100 times more images can be taken from the same sub-area, for example to observe a dynamic process.

This in 8 Illustrated scanning fluorescence microscope 13 has the following differences from that in figure 5 illustrated scanning fluorescence microscope. The point detector 21 is arranged behind the scanning means 22 and 23 as seen from the sample 8 so that the scanning means unscan the fluorescent light 20 coming from the sample 8 towards the point detector 21 . The scanning means 22 and 23 are provided both for scanning the partial area to be scanned with the zero point of the fluorescence prevention light 2 and for basically arranging or relocating the partial area in the sample 8 to be scanned. 8 8 also shows a fluorescent light detector 28 placed in front of the scanning means 22 and 23 from the sample of FIG. However, this is not a point detector, but a camera 29, ie a flat detector. This detector 28 can be provided in addition to the point detector 21 or instead of the point detector 21, with a dichroic mirror 30 deflecting the fluorescent light 20 to the detector 28 being arranged either temporarily or permanently between the objective 45 and the scanning means 22 and 23.

Furthermore, in accordance with the scanning fluorescence microscope 13 8 a switch-off light source 31 is provided, which is assigned expansion optics 32 in order to use the zero point of the fluorescence prevention light before scanning the respective partial area to be scanned 2 off light 34 to provide. The turn-off light 34 is coupled in with the aid of a dichroic mirror 43, and its intensity distribution in the sample 8 is shaped with the beam-shaping means 33 in such a way that switchable luminescence markers contained in the sample 8 are switched to an inactive state in a partial area of the sample 8 adjacent to the partial area to be scanned switches. In this inactive state, the switchable luminescence markers in the sample 8 cannot be excited with the excitation light 1 to emit fluorescent light 20 . Correspondingly, there is no relevant risk of bleaching for the luminescence markers in their inactive state from the fluorescence prevention light 2 in the form of stimulation light. This is how it is with the scanning fluorescence microscope 13 according to 8 possible to scan a partial area of the sample 8 adjoining an already scanned partial area to be scanned with the zero point of the fluorescence prevention light 2 and thereby to register fluorescent light 20 from the sample 8 because the luminescence markers located there were not omitted when scanning the previously scanned partial area to be scanned, since they were in their anti-bleaching inactive state.

In order to be able to stimulate the luminescence markers in the adjoining partial area to be scanned with the excitation light 1 to emit fluorescent light 20, they must be in their active state again. For this purpose, a spontaneous return of the luminescence markers from their inactive state to their active state can be awaited. The scanning fluorescence microscope 13 according to 8 but also has an additional turn-on light source 35 with expansion optics 36 to direct turn-on light 37 via a dichroic mirror 44 onto the sample 8, with which the luminescence markers in the next partial area to be scanned are initially switched to their active state. The sub-area of the sample 8 covered by the turn-on light 37 can be larger than the sub-area to be scanned next because the luminescence markers outside of the sub-area to be scanned next are then converted to their inactive state with the turn-off light 34 . With the scanning fluorescence microscope 13, the sample 8 can therefore be scanned in two stages, namely in large steps with the partial area to be scanned and in small steps with the zero point of the fluorescence prevention light 2 within each partial area to be scanned.

When the luminescence markers in the sample 8 are switched on and/or off with the switch-on light 37 or the switch-off light 34 , various switchable luminescence markers are also excited to emit fluorescent light 20 . This fluorescent light 20 thus already provides information about the concentration of the luminescence markers in the respective sub-area of the sample 8. This information can be evaluated accordingly and used for a decision as to whether it is at all worthwhile to scan the next sub-area to be scanned with the zero point of the fluorescence prevention light 2 or Not. When it is not worthwhile, such scanning is not performed either, so as not to irradiate the sample 8 with the fluorescence preventing light 2 unnecessarily. In addition, the fluorescent light 20 registered when the luminescence markers are switched on or off can also be used to define an upper and/or lower limit value for the fluorescent light registered at the respective location of the zero point of the fluorescence prevention light 2 in the partial area to be scanned, when it is reached or If not reached, exposure of the sample 8 to the luminescence prevention light 2 and the excitation light 1 is terminated in the sense of a RESCue method.

ultimately shows 8 a controller 38 for the light sources 14 and 17, the switch-on light source 35, the switch-off light source 31 and the scanning means 22 and 23 of the scanning fluorescence microscope 13 in order to control them according to the method according to the invention.

9 12 shows a partial area 7 to be scanned and the intensity maximum 3 of the fluorescence prevention light running around it when the zero point of the fluorescence prevention light is in the center point 10 of the partial area 7 to be scanned. In 9 In addition, an annular adjoining partial area 39 is drawn in, in which the sample 8 is exposed to the switch-off light 34 before the partial area 7 is scanned, in order to convert the switchable luminescence markers located here into their inactive state. The sub-area 39 leaves out the sub-area 7, ie in the sub-area 7 the intensity of the switch-off light 34 is zero or at least so small that it is not sufficient to switch off the luminescence marker within the time for which the switch-off light 34 is directed onto the sample 8 . In order to ensure that the switchable luminescence markers are in their active state at least in the sub-area 7 of the sample 8 to be scanned, the switch-on light 37 is switched on before and/or overlapping in time with the switch-off light 34 over a circular sub-area 40, which comprises the sub-area 7 to be scanned. aimed at sample 8.

10 illustrates how the sample 8 can be scanned with the partial area 7 to be scanned. while showing Figure 10(a) several successive layers of the circular portion 7 according to 9 in the sample 8 and in one of these partial areas 7 the path 9, along which a square area of the sample 8 is scanned within the partial area 7. Figure 10(b) shows how the sample 8 can be completely covered with such square partial areas 41 and correspondingly completely imaged.

11(a) 12 illustrates another form of scanning the sample 8 with the portion 7. Here the successive positions of the circular portion 7 are provided in the sample 8 in a hexagonal array. In this case, the path 9 extends, along which each partial area 7 with the zero point of the luminescence prevention light is sampled, over a regular hexagon. Figure 11(b) shows how the entire sample 8 is recorded and correspondingly imaged with these regular hexagons 42 .

In the 12 The embodiment of the method according to the invention shown in a block diagram begins with the marking 46 of a plurality of objects of interest with luminescence markers. In this case, each object of interest is indeed marked with several luminescence markers. However, the number of luminescence markers per object can be small and, for example, only two. In concrete terms, two different areas of the object of interest, such as a protein, can be marked with two identical or two different fluorescent markers in order to map the structure of the object in terms of how far these two areas of the object of interest are currently apart from one another. During the subsequent arrangement 47 of the plurality of objects of interest in the sub-areas 7 of a sample 8 to be scanned, one of these objects is arranged in each of the sub-areas 7 . This arrangement 47 of precisely one object of interest in each sub-area can be based on an immune reaction, via which an object of interest is linked to the center point of the respective sub-area 7 to be scanned. The subsequent determination 48 of the locations of the luminescence markers in the sample 8 is carried out by scanning the partial areas 7 to be scanned with the zero point of the intensity distribution of the light that affects the emission of luminescence light through the luminescence markers, ie for example luminescence excitation light. Specifically, the locations of the luminescence markers in the sample 8 can be determined from the intensities of the luminescence light that have been registered for the individual locations of the zero point in the sample 8 in relation to each of the partial areas 7 . If subsequently there is a change 49 in the environmental conditions of the objects of interest, which affects the structure of the objects and thus the relative locations of the luminescence markers in the sample 8, this effect can be detected by repeating the determination 48. In this case, the determination 48 can be repeated several times, with these repetitions already beginning during the change 49 and also being able to continue thereafter, in order to record the dynamics of the reaction of the objects of interest to the changed environmental conditions.

13 shows a sample 8 in which a plurality of partial areas 7 to be scanned are arranged in a defined pattern 51 opposite fixed points 50 . The pattern 51 is a square pattern in which the partial areas 7 are regularly arranged in rows 52 and columns 53 . Each of the partial areas 7 to be scanned can be found using the fixed points 50 and approached with the zero point of the intensity distribution of the light affecting the emission of luminescent light by the luminescent markers. The objects of interest can be coupled in centers 54 of the partial areas 7 by the already mentioned immune reaction, so that an object of interest is arranged overlapping each of the partial areas 7 .

14 12 shows an object 55 of interest in a highly schematic manner, which is marked in two areas with two luminescence markers 56 and 57, which are shown differently here. What is shown is that the luminescence markers 56 and 57 are arranged at the two ends of the linear object 55 . 14a shows a stretched structure of the object 55 while Figure 14b a curved or folded structure of the object 55 shows. The change between the stretched structure according to 14a and according to the folded structure Figure 14b can be the reaction of the object 55 to changed environmental conditions, for example a changed pH value or the presence of a specific chemical substance. The change in the structure can be easily registered with the method according to the invention by detecting the distance between the two luminescence markers 56 and 57 . For this purpose, the positions of the two luminescence markers 56 and 57 in the sample can be determined simultaneously if the luminescence light they emit can be separated due to different wavelengths or in terms of time because the two luminescence markers 56 and 57 only temporarily emit luminescence light. A temporary emission of luminescent light by only one of the two luminescent markers 56 and 57, even if they are identical, can be achieved by various circumstances, for example by the luminescent markers 56 and 57 temporarily entering a dark state as a result of their excitation with luminescent excitation light or as switchable ones Luminescence markers are formed, which can be temporarily switched to a dark state with additional turn-off light.

15 outlines four locations 58 and 59 for a partial area 7 to be scanned, at which the zero point of the intensity distribution of the light affecting the emission of luminescence light can be arranged in order to determine the location of a single emitting luminescence marker 56, 57 in the partial area 7. The location 58 is at the center 54 of the sub-area 7, and the locations 59 are on a circular arc in the edge area of the sub-area 7. By scanning the sub-area 7 with only four locations of the zero point, the location of the respective luminescence marker 56 or 57 in a plane of the sample, this determination can be done very quickly. If, in addition, the light that affects the emission of luminescence light and that is directed onto the sample with the intensity distribution with the zero point is excitation light, i.e. that only hits the respective luminescence marker 56, 57 with low intensity, only very few photons are produced required by the respective luminescence marker 56, 57 to determine its position in the partial area 7 to determine. Even with sensitive luminescence markers, this allows a repeated position determination in order to detect reactions of the object 55 of interest to changing environmental conditions.

REFERENCE LIST

1

excitation light

2

fluorescence prevention light

3

maximum intensity

4

zero

5

area

6

excitation

7

part to be scanned

8th

sample

9

Rail

10

Focus

11

dashed line

12

spiral track

13

scanning fluorescence microscope

14

light source

15

expansion optics

16

phase plate

17

another light source

18

expansion optics

19

dichroic mirror

20

fluorescent light

21

point detector

22

scanning means

23

scanning means

24

sample holder

25

shift icon

26

dichroic mirror

27

maximum intensity

28

detector

29

camera

30

dichroic mirror

31

off light source

32

expansion optics

33

beam shaping agent

34

off light

35

turn-on light source

36

expansion optics

37

turn on light

38

steering

39

adjoining section

40

subarea

41

square

42

hexagon

43

dichroic mirror

44

dichroic mirror

45

lens

46

mark (step)

47

arrange (step)

48

determine (step)

49

change (step)

50

fixed point

51

sample

52

Row

53

Split

54

Focus

55

object

56

luminescence marker

57

luminescence marker

58

location

59

location

f

focal point

I

intensity

D0

Distance

Claims (18)

1. Method of high-resolution imaging of a structure marked with luminescence markers (56, 57) in a sample (8),

   - wherein light, which has an effect on the emission of luminescence light by the luminescence markers (56, 57), is directed onto the sample (8) with an intensity distribution having a zero point (4) neighboured by intensity maxima (3),

   - wherein partial areas (7) to be scanned of the sample (8) are scanned with the zero point (4), and

   - wherein luminescence light emitted out of the area of the zero point (4) is registered and assigned to the location of the zero point (4) in the sample (8),

   characterised in

   - that several copies of an object (55) of interest are each arranged overlapping with one of the partial areas (7) to be scanned of the sample (8),

   - that the several copies of the object (55) of interest are subjected to varied surrounding conditions to monitor reactions of the object (55) of interest resulting therefrom, wherein the individual partial areas of the sample (8) are scanned during, and/or prior to and after the varied surrounding conditions with the respective zero point (4),

   - that dimensions of the partial areas (7) to be scanned of the sample (8) are delimited in at least one direction, in which the intensity maxima (3) are neighbouring the zero point (4) in the sample, such that they are not larger than 75 % of a distance (D₀) of the intensity maxima (3) in this direction,

   - that the light, which has an effect on the emission of luminescence light by the luminescence markers (56, 57), is

   - luminescence enabling light,

   - luminescence inhibiting light, which inhibits the emission of luminescence light by the luminescence markers (56, 57) outside the zero point (4), and

   - that, prior to scanning the partial area (7) to be scanned of the sample (8) with the zero point (4), switch-off light (34) is additionally directed onto the sample (8) with such an intensity distribution that, in a partial area (39) of the sample (8) neighbouring the partial area (7) to be scanned, it switches off the luminescence markers, which are made as switchable luminescence markers, into an inactive state, wherein the neighbouring partial area (39) is neighbouring the partial area (7) to be scanned in the at least one direction in which the intensity maxima (3) are neighbouring the zero point (4) in the sample.
2. Method of claim 1, characterised in that different subsets of the several copies of the object (55) of interest in the sample (8) are subjected to different varied surrounding conditions.
3. Method of claim 1 or 2, characterised in that the surrounding conditions are varied by adding a chemical substance.
4. Method of any of the preceding claims, characterised in that the several copies of the object (55) of interest are arranged in a pattern (51) defined with regard to fixed points (50) of the sample (8), and that the partial areas to be scanned of the sample (8) are approached with the zero point (4) relative to the fixed points (50) of the sample (8).
5. Method of any of the preceding claims, characterised in that the partial areas (7) to be scanned of the sample (8) are repeatedly scanned with the zero point (4), wherein, optionally, at least in one repetition of the scanning of the partial areas to be scanned of the sample (8), the zero point (4) is arranged at not more than 3n or 2n locations per partial area (7) to be scanned, wherein n is the number of the spatial directions in which the partial areas to be scanned of the sample (8) are scanned.
6. Method of any of the preceding claims, characterised in that the object (55) which is arranged in the sample (8) in several copies is selected from the group which includes molecules, proteins, complexes, synapses, membranes, cell components and viruses.
7. Method of any of the preceding claims, characterised in that the dimensions of the partial area (7) to be scanned of the sample (8), in the at least one direction, in which the intensity maxima (3) are neighbouring the zero point (4) in the sample, are

   - not larger than 50 %, 45 %, 25 % or 10 % of the distance (D₀) of the intensity maxima (3) in this direction, and/or

   - not larger than a way over which the intensity (I) of the light starting from the zero point (4) in this direction increases up to 50 %, 25 %, 10 % or 5 % of the intensity (I) of the light in the neighbouring intensity maxima (3).
8. Method of any of the preceding claims, characterised in that, prior to scanning the partial area (7) to be scanned of the sample (8), the structure in the sample (8) is imaged in another way to determine the position of the partial area (7) to be scanned in the sample (8), wherein, optionally, prior to scanning the partial area (7) to be scanned of the sample (8), a larger partial area of the sample (8) is scanned with the zero point (4) at an intensity of the light reduced by at least 50 % and/or at a scanning speed increased by at least 50 %.
9. Method of claim 8, characterised in that other scanning means are used for scanning the larger area of the sample (8) than for scanning the partial area (7) to be scanned of the sample (8), wherein, optionally,

   - for scanning the larger area of the sample (8) in at least one direction, a sample holder (24) is moved relative to an objective lens (17), by which the light is directed onto the sample (8), and/or

   - an electro-optical scanner, an acusto-optical deflector or a galvo mirror is used for scanning the partial area (7) to be scanned of the sample (8) in at least one direction.
10. Method of any of the preceding claims, characterised in that, in case that the light, which has an effect on the emission of luminescence light by the luminescence markers (56, 57), is luminescence enabling light, the luminescence enabling light, enables the emission of luminescence light by the luminescence markers (56, 57) in that it excites the luminescence markers (56, 57) for luminescence and/or transfers the luminescence markers (56, 57) out of a dark state into an excitable state.
11. Method of any of the claims 1 to 9, characterised in that, in caser that the light, which has an effect on the emission of luminescence light by the luminescence markers (56, 57), is luminescence inhibiting light, which inhibits the emission of luminescence light by the luminescence markers (56, 57) outside the zero point (4), the luminescence inhibiting light is stimulation light, which inhibits the emission of luminescence light by the luminescence markers (56, 57) outside the zero point (4) by stimulated emission, wherein the stimulation light is directed onto the sample (8) together with excitation light (1), which excites the luminescence markers (56, 57) for emission of luminescence light and which has an intensity distribution comprising a maximum (27) in the area of the zero point (4) of the luminescence inhibiting light.
12. Method of any of the preceding claims, characterised in that the sample is scanned with the partial area (7) to be scanned, wherein the partial area (7) to be scanned is scanned with the zero point (4) at all or at selected positions of the partial area (7) to be scanned.
13. Method of any of the preceding claims, characterised in that the intensity distribution of the switch-off light (34) has a local intensity minimum in the partial area (7) to be scanned, which is formed by destructive interference.
14. Method of any of the preceding claims, characterised in that prior to and/or temporarily overlapping with the directing of the switch-off lights (34) onto sample (8), switch-on light (37) is directed onto the partial area (7) to be scanned of the sample (8), which switches the switchable luminescence markers (56, 57) into their active state.
15. Method of any of the preceding claims, characterised in that luminescence light (20) emitted in switching on and/or switching off of the switchable luminescence markers (56, 57) is registered and evaluated, wherein the result of the evaluation, optionally, is

    - whether the partial area (7) to be scanned and neighboured by the neighbouring partial area (39), is scanned with the zero point (4), and/or

    - whether the excitation light (1) is directed onto the sample (8) in the partial area (7) to be scanned and neighboured by the neighbouring partial area (39), and/or

    - under which conditions the directing of the stimulation light onto the sample (8) in each position of the zero point (4) in the partial area (7) to be scanned and neighboured by the neighbouring partial area (39), and the registering of the luminescence light (20) emitted out of the area of the zero point (4) are terminated.
16. Method of any of the preceding claims, characterised in that luminescence light emitted out of the area of the zero point (4) is registered with a point sensor (21), whose location with regard to the sample (8) is not altered in scanning the partial area (7) to be scanned of the sample (8).
17. Method of any of the preceding claims, characterised in that the several partial areas (7) to be scanned of the sample (8) are scanned simultaneously.
18. Method of high-resolution imaging of a structure marked with luminescence markers (56, 57) in a sample (8),

    - wherein light, which has an effect on the emission of luminescence light by the luminescence markers (56, 57), is directed onto the sample (8) with an intensity distribution having a zero point (4) neighboured by intensity maxima (3),

    - wherein partial areas (7) to be scanned of the sample (8) are scanned with the zero point (4), and

    - wherein luminescence light emitted out of the area of the zero point (4) is registered and assigned to the location of the zero point (4) in the sample (8),

    - wherein several copies of an object (55) of interest are each arranged overlapping with one of the partial areas (7) to be scanned of the sample (8),

    - wherein the several copies of the object (55) of interest are subjected to varied surrounding conditions to monitor reactions of the object (55) of interest resulting therefrom, wherein the individual partial areas of the sample (8) are scanned during, and/or prior to and after the varied surrounding conditions with the respective zero point (4),

    - wherein dimensions of the partial areas (7) to be scanned of the sample (8) are delimited in at least one direction, in which the intensity maxima (3) are neighbouring the zero point (4) in the sample, such that they are not larger than 75 % of a distance (D₀) of the intensity maxima (3) in this direction,

    - wherein the several copies of the object (55) of interest are arranged in a pattern (51) defined with regard to fixed points (50) of the sample (8), and

    - wherein the partial areas to be scanned of the sample (8) are approached with the zero point (4) relative to the fixed points (50) of the sample (8).

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